Non-growth substrate ethane perturbs core methanotrophy in obligate methanotroph OB3b upon nutrient availability.

UNLABELLED

Obligate methanotrophs grow exclusively on C1 compounds; however, their widespread ecological distribution and ability to produce polyhydroxybutyrate (PHB) under nutrient stress suggest a greater-than-expected degree of metabolic flexibility. However, the metabolic responses of type II obligate methanotrophs to heterogeneous substrate conditions, common in natural and engineered environments, remain poorly understood. Here, we investigated how ethane (CH), a non-growth substrate and methane monooxygenase (MMO) co-substrate, affects the metabolism of OB3b under varying CH concentrations and nutrient availabilities (CH, O, and nitrogen). Exposure to CH impaired CH oxidation and cell growth while stimulating PHB synthesis, with these effects becoming more pronounced at higher CH levels. Externally supplemented reducing equivalents were preferentially directed toward CH uptake rather than CH oxidation under co-substrate conditions. Additionally, acetate derived from CH exerted divergent effects on carbon assimilation during growth and PHB accumulation phases, without directly interfering with CH oxidation. RT-qPCR unraveled that MMO gene expression responded to CH in a nutrient availability-dependent manner, with opposing patterns during growth and PHB accumulation phases. Interestingly, MMO transcript levels did not correlate with CH oxidation rates, suggesting that MMO mRNA abundance may not serve as a reliable proxy for actual methanotrophic activity under co-substrate conditions. These findings highlight the significant effects of heterogeneous substrate environments-containing co-metabolic substrates and microbial metabolites-on obligate methanotrophs, offering insights for revisiting methanotrophic activities in ecosystems and optimizing biotechnological applications.

IMPORTANCE

Type II methanotrophs present a dual advantage: mitigating methane emissions and producing bioproducts such as polyhydroxybutyrate (PHB). However, their full potential remains untapped, partly due to a limited understanding of how co-occurring gases influence their metabolism. Methane-rich emissions from both natural and anthropogenic sources are frequently accompanied by secondary gases, such as ethane, which create heterogeneous substrate conditions. This study reveals that ethane, a non-growth co-metabolic substrate, significantly modulates the metabolism of type II obligate methanotrophs, affecting microbial growth, methane oxidation, and PHB synthesis. These results advance our understanding of the metabolic plasticity of these organisms and also reveal new opportunities to leverage secondary substrates for selectively fine-tuning beneficial methanotrophic activities, such as biopolymer production.